Gene-expression reporter assays bridge bioinformatics data with gene-specific bioassays to monitor effects of ligands, compounds, antibodies, siRNAs, miRNAs, and peptides on gene expression. In cell culture, they provide for bench top or high throughput screening applicable to gene regulation, pathway discovery, target validation, drug discovery, and cellular target efficacy. Used in vivo with whole animal imaging, they provide for temporal and quantitative analysis of drug delivery and in vivo target efficacy. The most commonly used reporter-gene assays place an easily monitored reporter gene such as a fluorescent protein or luciferase downstream to cloned promoter and transcriptional regulatory elements. In an exogenous reporter, the vector is introduced into recipient cells where expression proceeds either transiently, or after random and stable integration into the genome. Exogenous reporter-gene assays are highly unpredictable, and often incapable of indexing gene expression to known regulatory agents. They can provide erroneous data and mislead research investigations, thereby costing time, expense and unrealized discovery and development opportunities. In contrast, in an endogenous reporter, reporter genes are introduced at their normal chromosomal locus where native promoters, regulatory elements, local chromosomal modifications and micro-RNA regulatory sequences are utilized to reliably and accurately index gene regulation. However, very few endogenous human gene- expression reporter cell assays are available and sensitivity limits accurate monitoring of many genes. Compounding this, current gene trapping and gene targeting methods for producing assays require large investments in resources, time, and cost, making them unattainable for most laboratories. In phase I studies, we propose to combine Xactagen's prior research advances in reporter cell sensitivity with innovative methods to mass produce, store and retrieve endogenous, human gene expression reporter cells. Our hypothesis is that we will have extensive gene coverage, sensitivity to monitor expression of even poorly expressed genes, reliable responses to known gene regulators, and the ability to retrieve specific reporter cells from our libraries for delivery to researchers within 4-6 weeks of ordering. Our specific aims are: (1) Generate a pilot library of 19,200 arrayed gene-trapped reporter cells (NIH-H292 human mucoepidermoid carcinoma cell line), (2) Identify vector insertion sites, and characterize gene/transcription unit representation, and (3) Retrieve 10 endogenous reporter cells and verify functionality. This innovative research provides for cost-effective and highly expedited production of human endogenous gene expression reporter cell libraries in weeks as opposed to years. As a result, more researchers will have access to endogenous human reporter cells and will be able to focus on cell culture and in vivo applications, rather than on production. Additionally, researchers can avoid exogenous reporter cells with their unpredictable indexing of gene expression. Ultimately, scientists will make more viable research and medical discoveries in less time and at far less cost. PUBLIC HEALTH RELEVANCE: Current methods for production of endogenous gene expression reporter cells are laborious and cost- prohibitive. The ultimate goal of this research i to provide cost-effective and highly expedited production of human endogenous reporter cell libraries - in weeks as opposed to years. Hundreds of thousands of readily available, highly sensitive, easy to use, and affordable gene expression reporter cells produced in diverse human cell lines and stem cells are expected to increase productivity of scientific research and drug discovery.